Cell death induced by oxidative stress occurs during ischemia/reperfusion, leading to failure of different organs such as the heart, brain, liver, and/or kidneys. Furthermore, oxidative stress is often the mediator of drug- and toxicant-induced cell death. Our previous studies in renal cells suggested that inhibition of calcium- independent phospholipase A2 (iPLA2) with the iPLA2 inhibitor bromoenol lactone (BEL) potentiated lipid peroxidation and necrotic cell death induced by oxidants.These results led us to the hypothesis that iPLA2 acts to repair or prevent lipid peroxidation induced by oxidants. We have identified IPLA2 gamma (Group VIB) in renal cells and localized it to the endoplasmic reticulum (ER). Exciting new preliminary studies have revealed that iPLA2 gamma also is present in the mitochondria of renal cells. The localization of a protein to both the ER and mitochondria is rare and limited to few other proteins. It should be noted that the major sources of reactive oxygen species in cells, ER and mitochondria, coincide with IPLA2 gamma localization. Our current hypothesis is that iPLA2 gamma protects cells from oxidative stress by preserving endoplasmic reticulum and mitochondrial membrane integrity and function. We propose to investigate this hypothesis through the following Specific Aims: Specific Aim 1. Determine the mechanism by which iPLA2 gamma is targeted to the ER and mitochondria. Specific Aim 2. Determine the consequences of "over-expression"and "knock-down" of iPLA2 gamma on ER and mitochondrial functions and necrotic cell death following oxidative stress. Specific Aim 3. Determine the mechanism(s) by which iPLA gamma protects ER and mitochondria during oxidative stress. Successful completion of these aims will increase our limited knowledge on the targeting of proteins to multiple cellular locations and elucidate the function of iPLA2 gamma, a potential natural defense enzyme against oxidative stress in the mitochondria and ER. Ultimately, these studies may lead to new therapeutic and pharmacological approaches to increase cell and organ survival in numerous pathologic situations.